New insights into the lymphovascular microanatomy of the colon and the risk of metastases in pT1 colorectal cancer obtained with quantitative methods and three-dimensional digital reconstruction

Aims: UK faecal occult blood test screening has tripled the proportion of pT1 colorectal cancers. The risk of metastasis is predicted by depth of invasion, suggesting that access to deep lymphovascular vessels is important. The aim of this study was to quantify the distribution and size of the submucosal vasculature, and generate a novel three-dimensional (3D) model to validate the findings. Methods and results: Thirty samples of normal large bowel wall were immunostained with CD31, a vascular endothelium marker, to identify blood vessels, which were quantified and digitally analysed for their number, circumference, area and diameter in the deep mucosa and submucosa (Sm1, Sm2, and Sm3). The model required serial sections, a double immunostain (using CD31 and D2-40), and 3D reconstruction. Significant differences were shown between submucosal layers in the number, circumference and area of vessels (P < 0.001). Blood vessels were most numerous in the mucosa (11.79 vessels/0.2 mm2) but smaller [median area of 247 μm2, interquartile range (IQR) 162–373 μm2] than in Sm2, where they were fewer in number (6.92 vessels/0.2 mm2) but considerably larger (2086 μm2, IQR 1007–4784 μm2). The 3D model generated novel observations on lymphovascular structures. Conclusions: The number and size of blood vessels do not increase with depth of submucosa, as hypothesized. The distribution of vessels suggests that we should investigate the area or volume of submucosal invasion rather than the depth.